Methods and compositions for efficient nucleic acid sequencing

ABSTRACT

Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

This is a continuation of U.S. application Ser. No. 09/272,232, filedMar. 18, 1999, (now U.S. Pat. No. 6,401,267, issued Jun. 11, 2002) whichin turn is a continuation of U.S. application Ser. No. 08/619,649 filedMar. 27, 1996, which is a 371 nationalization application of Ser. No.PCT/US94/10945, filed Sep. 27, 1994, which in turn is acontinuation-in-part of U.S. application Ser. No. 08/303,058 filed Sep.8, 1994 (now abandoned), which in turn is a continuation-in-part of U.S.application Ser. No. 08/127,420 filed Sep. 27, 1993 (now abandoned); theentire text and figures of which disclosures are specificallyincorporated herein by reference without disclaimer.

The U.S. Government owns rights in the present invention pursuant toDepartment of Energy grant LDRD 03235 and Contract No. W-31-109-ENG-38between the U.S. Department of Energy and the University of Chicago,representing Argonne National Laboratory.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to the field of molecularbiology. The invention particularly provides novel methods andcompositions to enable highly efficient sequencing of nucleic acidmolecules. The methods of the invention are suitable for sequencing longnucleic acid molecules, including chromosomes and RNA, without cloningor subcloning steps.

2. Description of the Related Art

Nucleic acid sequencing forms an integral part of scientific progresstoday. Determining the sequence, i.e. the primary structure, of nucleicacid molecules and segments is important in regard to individualprojects investigating a range of particular target areas. Informationgained from sequencing impacts science, medicine, agriculture and allareas of biotechnology. Nucleic acid sequencing is, of course, vital tothe human genome project and other large-scale undertakings, the aim ofwhich is to further our understanding of evolution and the function oforganisms and to provide an insight into the causes of various diseasestates.

The utility of nucleic acid sequencing is evident, for example, theHuman Genome Project (HGP), a multinational effort devoted to sequencingthe entire human genome, is in progress at various centers. However,progress in this area is generally both slow and costly. Nucleic acidsequencing is usually determined on polyacrylamide gels that separateDNA fragments in the range of 1 to 500 bp, differing in length by onenucleotide. The actual determination of the sequence, i.e., the order ofthe individual A, G, C and T nucleotides may be achieved in two ways.Firstly, using the Maxam and Gilbert method of chemically degrading theDNA fragment at specific nucleotides (Maxam & Gilbert, 1977), orsecondly, using the dideoxy chain termination sequencing methoddescribed by Sanger and colleagues (Sanger et al., 1977). Both methodsare time-consuming and laborious.

More recently, other methods of nucleic acid sequencing have beenproposed that do not employ an electrophoresis step, these methods maybe collectively termed Sequencing By Hybridization or SBH (Drmanac etal., 1991; Cantor et al., 1992; Drmanac & Crkvenjakov, U.S. Pat. No.5,202,231). Development of certain of these methods has given rise tonew solid support type sequencing tools known as sequencing chips. Theutility of SBH in general is evidenced by the fact that U.S. Patentshave been granted on this technology. However, although SBH has thepotential for increasing the speed with which nucleic acids can besequenced, all current SBH methods still suffer from several drawbacks.

SBH can be conducted in two basic ways, often-referred to as Format 1and Format 2 (Cantor et al., 1992). In Format 1, oligonucleotides ofunknown sequence, generally of about 100-1000 nucleotides in length, arearrayed on a solid support or filter so that the unknown samplesthemselves are immobilized (Strezoska et al., 1991; Drmanac &Crkvenjakov, U.S. Pat. No. 5,202,231). Replicas of the array are theninterrogated by hybridization with sets of labeled probes of about 6 to8 residues in length. In Format 2, a sequencing chip is formed from anarray of oligonucleotides with known sequences of about 6 to 8 residuesin length (Southern, WO 89/10977; Khrapko et al., 1991; Southern et al.,1992). The nucleic acids of unknown sequence are then labeled andallowed to hybridize to the immobilized oligos.

Unfortunately, both of these SBH formats have several limitations,particularly the requirement for prior DNA cloning steps. In Format 1,other significant problems include attaching the various nucleic acidpieces to be sequenced to the solid surface support or preparing a largeset of longer probes. In Format 2, major problems include labelling thenucleic acids of unknown sequence, high noise to signal ratios thatgenerally result, and the fact that only short sequences can bedetermined. Further problems of Format 2 include the secondary structureformation that prevents access to some targets and the differentconditions that are necessary for probes with different GC contents.Therefore, the art would clearly benefit from a new procedure fornucleic acid sequencing, and particularly, one that avoids the tediousprocesses of cloning and/or subcloning.

SUMMARY OF THE INVENTION

The present invention seeks to overcome these and other drawbacksinherent in the prior art by providing new methods and compositions forthe sequencing of nucleic acids. The novel techniques described hereinhave been generally termed Format 3 by the inventors and representmarked improvements over the existing Format 1 and Format 2 SBH methods.In the Format 3 sequencing provided by the invention, nucleic acidsequences are determined by means of hybridization with two sets ofsmall oligonucleotide probes of known sequences. The methods of theinvention allow high discriminatory sequencing of extremely largenucleic acid molecules, including chromosomal material or RNA, withoutprior cloning, subcloning or amplification. Furthermore, the presentmethods do not require large numbers of probes, the complex synthesis oflonger probes, or the labelling of a complex mixture of nucleic acidssegments.

To determine the sequence of a nucleic acid according to the methods ofthe present invention, one would generally identify sequences from thenucleic acid by hybridizing with complementary sequences from two setsof small oligonucleotide probes (oligos) of defined length and knownsequence, which cover most combinations of sequences for that length ofprobe. One would then analyze the sequences identified to determinestretches of the identified sequences that overlap, and reconstruct orassemble the complete nucleic acid sequence from such overlappingsequences.

The sequencing methods may be conducted using sequential hybridizationwith complementary sequences from the two sets of small oligos.Alternatively, a mode described as “cycling” may be employed, in whichthe two sets of small oligos are hybridized with the unknown sequencessimultaneously. The term “cycling” is applied as the discriminatory partof the technique comes from then increasing the temperature to “melt”those hybrids that are non-complementary. Such cycling techniques arecommonly employed in other areas of molecular biology, such as PCR, andwill be readily understood by those of skill in the art in light whenreading the present disclosure.

The invention is applicable to sequencing nucleic acid molecules of verylong length. As a practical matter, the nucleic acid molecule to besequenced will generally be fragmented to provide small or intermediatelength nucleic acid fragments that may be readily manipulated. The termnucleic acid fragment, as used herein, most generally means a nucleicacid molecule of between about 10 base pairs (bp) and about 100 bp inlength. The most preferred methods of the invention are contemplated tobe those in which the nucleic acid molecule to be sequenced is treatedto provide nucleic acid fragments of intermediate length, i.e., ofbetween about 10 bp and about 40 bp. However, it should be stressed thatthe present invention is not a method of completely sequencing smallnucleic acid fragments, rather it is a method of sequencing nucleic acidmolecules per se, which involves determining portions of sequence fromwithin the molecule—whether this is done using the whole molecule, orfor simplicity, whether this is achieved by first fragmenting themolecule into smaller sized sections of from about 4 to about 1000bases.

Sequences from nucleic acid molecules are determined by hybridizing tosmall oligonucleotide probes of known sequence. In referring to “smalloligonucleotide probes”, the term “small” means probes of less than 10bp in length, and preferably, probes of between about 4 bp and about 9bp in length. In one exemplary sequencing embodiment, probes of about 6bp in length are contemplated to be particularly useful. For the sets ofoligos to cover all combinations of sequences for the length of probechosen, their number will be represented by 4^(F), wherein F is thelength of the probe. For example, for a 4-mer, the set would contain 256probes; for a 5-mer, the set would contain 1024 probes; for a 6-mer,4096 probes; a 7-mer, 16384 probes; and the like. The synthesis ofoligos of this length is very routine in the art and may be achieved byautomated synthesis.

In the methods of the invention, one set of the small oligonucleotideprobes of known sequence, which may be termed the first set, will beattached to a solid support, i.e., immobilized on that support in such away so that they are available to take part in hybridization reactions.The other set of small oligonucleotide probes of known sequence, whichmay be termed the second set, will be probes that are in solution andthat are labelled with a detectable label. The sets of oligos mayinclude probes of the same or different lengths.

The process of sequential hybridization means that nucleic acidmolecules, or fragments, of unknown sequence can be hybridized to thedistinct sets of oligonucleotide probes of known sequences at separatetimes (FIG. 1). The nucleic acid molecules or fragments will generallybe denatured, allowing hybridization, and added to the first,immobilized set of probes under discriminating hybridization conditionsto ensure that only fragments with complementary sequences hybridize.Fragments with non-complementary sequences are removed and the nextround of discriminating hybridization is then conducted by adding thesecond, labelled set of probes, in solution, to the combination offragments and probes already formed. Labelled probes that hybridizeadjacent to a fixed probe will remain attached to the support and can bedetected, which is not the case when there is space between the fixedand labelled probes (FIG. 1).

The process of simultaneous hybridization means that the unknownsequence nucleic acid molecules can be contacted with the distinct setsof oligonucleotide probes of known sequences at the same time.Hybridization will occur under discriminating hybridization conditions.Fragments with non-complementary sequences are then “melted”, i.e.,removed by increasing the temperature, and the next round ofdiscriminating hybridization is then conducted, allowing anycomplementary second probes to hybridize. Labelled probes that hybridizeadjacent to a fixed probe will then be detected in the same manner.

Nucleic acid sequences that are “complementary” are those that arecapable of base-pairing according to the standard Watson-Crickcomplementarity rules, and variations of the rules as they apply tomodified bases. That is, that the larger purines, or modified purines,will always base pair with the smaller pyrimidines to form only knowncombinations. These include the standard paris of guanine paired withCytosine (G:C) and Adenine paired with either Thymine (A:T), in the caseof DNA, or Adenine paired with Uracil (A:U) in the case of RNA. The useof modified bases, or the so-called Universal Base (M, Nichols et al.,1994) is also contemplated.

As used herein, the term “complementary sequences” means nucleic acidsequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, nucleic acid sequencesof six bases in length may be termed complementary when they hybridizeat five out of six positions with only a single mismatch. Naturally,nucleic acid sequences that are “completely complementary” will benucleic acid sequences that are entirely complementary throughout theirentire length and have no base mismatches.

After identifying, by hybridization to the oligos of known sequence,various individual sequences that are part of the nucleic acidfragments, these individual sequences are next analyzed to identifystretches of sequences that overlap. For example, portions of sequencesin which the 5′ end is the same as the 3′ end of another sequence, orvice versa, are identified. The complete sequence of the nucleic acidmolecule or fragment can then be delineated, i.e., it can bereconstructed from the overlapping sequences thus determined.

The processes of identifying overlapping sequences and reconstructingthe complete sequence will generally be achieved by computationalanalysis. For example, if a labelled probe 5′-TTTTTT-3′ hybridizes tothe spot containing the fixed probe 5′-AAAAAA-3′, a 12-mer sequence fromwithin the nucleic acid molecule is defined, namely 5′-AAAAAATTTTTT-3′(SEQ ID NO:1), i.e. the sequence of the two hybridized probes iscombined to reveal a previously unknown sequence. The next question tobe answered is which nucleotide follows next after the newly determined5′ AAAAAATTTTTT-3′ (SEQ ID NO:1) sequence. There are four possibilitiesrepresented by the fixed probe 5′-AAAAAT-3′ and labelled probes5′-TTTTTA-3′ for A; 5′-TTTTTT-3′ for T; 5′-TTTTTC-3′ for C; and5′-TTTTTG-3′ for G. If, for example, the probe 5′-TTTTTC-3′ is positiveand the other three are negative, then the assembled sequence isextended to 5′-AAAAAATTTTTTC-3′ (SEQ ID NO:2). In the next step, analgorithm determines which of the labelled probes TTTTCA, TTTTCT, TTTTCCor TTTTCG are positive at the spot containing the fixed probe AAAATT.The process is repeated until all positive (F+P) oligonucleotidesequences are used or defined as false positives.

The present invention thus provides a very effective way to sequencenucleic acid fragments and molecules of long length. Large nucleic acidmolecules, as defined herein, are those molecules that need to befragmented prior to sequencing. They will generally be of at least about45 or 50 base pairs (bp) in length, and will most often be longer. Infact, the methods of the invention may be used to sequence nucleic acidmolecules with virtually no upper limit on length, so that sequences ofabout 100 bp, 1 kilobase (kb), 100 kb, 1 megabase (Mb), and 50 Mb ormore may be sequenced, up to and including complete chromosomes, such ashuman chromosomes, which are about 100 Mb in length. Such a large numberis well within the scope of the present invention and sequencing thisnumber of bases will require two sets of 8-mers or 9-mers (so thatF+P≈16-18). The nucleic acids to be sequenced may be DNA, such as cDNA,genomic DNA, microdissected chromosome bands, cosmid DNA or YAC inserts,or may be RNA, including mRNA, rRNA, tRNA or snRNA.

The process of determining the sequence of a long nucleic acid moleculeinvolves simply identifying sequences of length F+P from the moleculeand combining the sequences using a suitable algorithm. In practicalterms, one would most likely first fragment the nucleic acid molecule tobe sequenced to produce smaller fragments, such as intermediate lengthnucleic acid fragments. One would then identify sequences of length F+Pby hybridizing, e.g., sequentially hybridizing, the fragments tocomplementary sequences from the two sets of small oligonucleotideprobes of known sequence, as described above. In this manner, thecomplete nucleic acid sequence of extremely large molecules can bereconstructed from overlapping sequences of length F+P.

Whether the nucleic acid to be sequenced is itself an intermediatelength fragment or is first treated to generate such length fragments,the process of identifying sequences from such nucleic acid fragments byhybridizing to two sets of small oligonucleotide probes of knownsequence is central to the sequencing methods disclosed herein. Thisprocess generally comprises the following steps:

-   -   (a) contacting the set or array of attached or immobilized        oligonucleotide probes with the nucleic acid fragments under        hybridization conditions effective to allow fragments with a        complementary sequence to hybridize sufficiently to a probe,        thereby forming primary complexes wherein the fragment has both        hybridized and non-hybridized, or “free”, sequences;    -   (b) contacting the primary complexes with the set of labelled        oligonucleotide probes in solution under hybridization        conditions effective to allow probes with complementary        sequences to hybridize to a non-hybridized or free fragment        sequence, thereby forming secondary complexes wherein the        fragment is hybridized to both an attached (immobilized) probe        and a labelled probe;    -   (c) removing from the secondary complexes any labelled probes        that have not hybridized adjacent to an attached probe, thereby        leaving only adjacent secondary complexes;    -   (d) detecting the adjacent secondary complexes by detecting the        presence of the label in the labelled probe; and    -   (e) identifying oligonucleotide sequences from the nucleic acid        fragments in the adjacent secondary complexes by combining or        connecting the known sequences of the hybridized attached and        labelled probes.

The hybridization or ‘washing conditions’ chosen to conduct either one,or both, of the hybridization steps may be manipulated according to theparticular sequencing embodiment chosen. For example, both of thehybridization conditions may be designed to allow oligonucleotide probesto hybridize to a given nucleic acid fragment when they containcomplementary sequences, i.e., substantially matching sequences, such asthose sequences that hybridize at five out of six positions. Thehybridization steps would preferably be conducted using a simple roboticdevice as is routinely used in current sequencing procedures.

Alternatively, the hybridization conditions may be designed to allowonly those oligonucleotide probes and fragments that have completelycomplementary sequences to hybridize. These more discriminating or‘stringent’ conditions may be used for both distinct steps of asequential hybridization process or for either step alone. In suchcases, the oligonucleotide probes, whether immobilized or labelledprobes, would only be allowed to hybridize to a given nucleic acidfragment when they shared completely complementary sequences with thefragment.

The hybridization conditions chosen will generally dictate the degree ofcomplexity required to analyze the data obtained. Equally, the computerprograms available to analyze any data generated may dictate thehybridization conditions that must be employed in a given laboratory.For example, in the most discriminating process, both hybridizationsteps would be conducted under conditions that allow only oligos andfragments with completely complementary sequences to hybridize. As therewill be no mismatched bases, this method involves the least complexcomputational analyses and, for this reason, it is the currentlypreferred method for practicing the invention. However, the use of lessdiscriminating conditions for one or both hybridization steps also fallswithin the scope of the present invention.

Suitable hybridization conditions for use in either or both steps may beroutinely determined by optimization procedures or ‘pilot studies’.Various types of pilot studies are routinely conducted by those skilledin the art of nucleic acid sequencing in establishing working proceduresand in adapting a procedure for use in a given laboratory. For example,conditions such as the temperature; the concentration of each of thecomponents; the length of time of the steps; the buffers used and theirpH and ionic strength may be varied and thereby optimized.

In preferred embodiments, the nucleic acid sequencing method of theinvention involves a discriminating step to select for secondaryhybridization complexes that include immediately adjacent immobilizedand labelled probes, as distinct from those that are not immediatelyadjacent and are separated by one, two or more bases. A variety ofprocesses are available for removing labelled probes that are nothybridized immediately adjacent to an attached probe, i.e., nothybridized back to back, each of which leaves only-the immediatelyadjacent secondary complexes.

Such discriminatory processes may rely solely on washing steps ofcontrolled stringency wherein the hybridization conditions employed aredesigned so that immediately adjacently probes remain hybridized due tothe increased stability afforded by the stacking interactions of theadjacent nucleotides. Again, washing conditions such as temperature,concentration, time, buffers, pH, ionic strength and the like, may bevaried to optimize the removal of labelled probes that are notimmediately adjacent.

In preferred embodiments the immediately adjacent immobilized andlabelled probes would be ligated, i.e., covalently joined, prior toperforming washing steps to remove any non-ligated probes. Ligation maybe achieved by treating with a solution containing a chemical ligatingagent, such as, e.g., water-soluble carbodiimide or cyanogen bromide.More preferably, a ligase enzyme, such as T₄ DNA ligase from T₄bacteriophage, which is commercially available from many sources (e.g.,Biolabs), may be employed. In any event, one would then be able toremove non-immediately adjacent labelled probes by more stringentwashing conditions that cannot affect the covalently connected labeledand fixed probes.

The remaining adjacent secondary complexes would be detected byobserving the location of the label from the labelled probes presentwithin the complexes. The oligonucleotide probes may be labeled with achemically-detectable label, such as fluorescent dyes, or adequatelymodified to be detected by a chemiluminescent developing procedure, orradioactive labels such as ³⁵S, ³H, ³²P or ³³P, with ³³P currently beingpreferred. Probes may also be labeled with non-radioactive isotopes anddetected by mass spectrometry.

Currently, the most preferred method contemplated for practicing thepresent invention involves performing the hybridization steps underconditions designed to allow only those oligonucleotide probes andfragments that have completely complementary sequences to hybridize andthat allow only those probes that are immediately adjacent to remainhybridized. This method subsequently requires the least complexcomputational analysis.

Where the nucleic acid molecule of unknown sequence is longer than about45 or 50 bp, one effective method for determining its sequence generallyinvolves treating the molecule to generate nucleic acid fragments ofintermediate length, and determining sequences from the fragments. Thenucleic acid molecule, whether it be DNA or RNA may be fragmented by anyone of a variety of methods including, for example, cutting byrestriction enzyme digestion, shearing by physical means such asultrasound treatment, by NaOH treatment or by low pressure shearing.

In certain embodiments, e.g., involving small oligonucleotide probesbetween about 4 bp and about 9 bp in length, one may aim to producenucleic acid fragments of between about 10 bp and about 40 bp in length.Naturally, longer length probes would generally be used in conjunctionwith sequencing longer length nucleic acid fragment, and vice versa. Incertain preferred embodiments, the small oligonucleotide probes usedwill be about 6 bp in length and the nucleic acid fragments to besequenced will generally be about 20 bp in length. If desired, fragmentsmay be separated by size to obtain those of an appropriate length, e.g.,fragments may be run on a gel, such as an agarose gel, and those withapproximately the desired length may be excised.

The method for determining the sequence of a nucleic acid molecule mayalso be exemplified using the following terms. Initially one wouldrandomly fragment an amount of the nucleic acid to be sequenced toprovide a mixture of nucleic acid fragments of length T. One wouldprepare an array of immobilized oligonucleotide probes of knownsequences and length F and a set of labelled oligonucleotide probes insolution of known sequences and length P, wherein F+P≦T and, preferably,wherein T≈3F.

One would then contact the array of immobilized oligonucleotide probeswith the mixture nucleic acid fragments under hybridization conditionseffective to allow the formation of primary complexes with hybridized,complementary sequences of length F and non-hybridized fragmentsequences of length T−F. Preferably, the hybridized sequences of lengthF would contain only completely complementary sequences.

The primary complexes would then be contacted with the set of labelledoligonucleotide probes under hybridization conditions effective to allowthe formation of secondary complexes with hybridized, complementarysequences of length F and adjacent hybridized, complementary sequencesof length P. In preferred embodiments, only those labelled probes withcompletely complementary sequences would be allowed to hybridize andonly those probes that hybridize immediately adjacent to an immobilizedprobe would be allowed to remain hybridized. In the most preferredembodiments, the adjacent immobilized and labelled oligonucleotideprobes would also be ligated at this stage.

Next one would detect the secondary complexes by detecting the presenceof the label and identify sequences of length F+P from the nucleic acidfragments in the secondary complexes by combining the known sequences ofthe hybridized immobilized and labelled probes. Stretches of thesequences of length F+P that overlap would then be identified, therebyallowing the complete nucleic acid sequence of the molecule to bereconstructed or assembled from the overlapping sequences determined.

In the methods of the invention, the oligonucleotides of the first setmay be attached to a solid support, i.e. immobilized, by any of themethods known to those of skill in the art. For example, attachment maybe via addressable laser-activated photodeprotection (Fodor et al.,1991; Pease et al., 1994). One generally preferred method is to attachthe oligos through the phosphate group using reagents such as nucleosidephosphoramidite or nucleoside hydrogen phosphorate, as described bySouthern & Maskos (PCT Patent Application WO 90/03382, incorporatedherein by reference), and using glass, nylon or teflon supports. Anotherpreferred method is that of light-generated synthesis described by Peaseet al. (1994; incorporated herein by reference). One may also purchasesupport bound oligonucleotide arrays, for example, as have been offeredfor sale by Affymetrix and Beckman.

The immobilized oligonucleotides may be formed into an array comprisingall probes or subsets of probes of a given length (preferably about 4 to10 bases), and more preferably, into multiple arrays of immobilizedoligonucleotides arranged to form a so-called “sequencing chip”. Oneexample of a chip is that where hydrophobic segments are used to createdistinct spatial areas. The sequencing chips may be designed fordifferent applications like mapping, partial sequencing, sequencing oftargeted regions for diagnostic purposes, mRNA sequencing and largescale genome sequencing. For each application, a specific chip may bedesigned with different sized probes or with an incomplete set ofprobes.

In one exemplary embodiment, both sets of oligonucleotide probes wouldbe probes of six bases in length, i.e., 6-mers. In this instance, eachset of oligos contains 4096 distinct probes. The first set probes ispreferably fixed in an array on a microchip, most conveniently arrangedin 64 rows and 64 columns. The second set of 4096 oligos would belabeled with a detectable label and dispensed into a set of distincttubes. In this example, 4096 of the chips would be combined in a largearray, or several arrays. After hybridizing the nucleic acid fragments,a small amount of the labeled oligonucleotides would be added to eachmicrochip for the second hybridization step, only one of each of the4096 nucleotides would be added to each microchip.

Further embodiments of the invention include kits for use in nucleicacid sequencing. Such kits will generally comprise a solid supporthaving attached an array of oligonucleotide probes of known sequences,as shown in FIG. 2A, FIG. 2B and FIG. 2C, wherein the oligonucleotidesare capable of taking part in hybridization reactions, and a set ofcontainers comprising solutions of labelled oligonucleotide probes ofknown sequences. Arrangements such as those shown in FIG. 4 are alsocontemplated. This depicts the use of the Universal Base, either as anattachment method, or at the terminus to give an added dimension to thehybridization of fragments.

In the kits, the attached oligonucleotide probes and those in solutionmay be between about 4 bp and about 9 bp in length, with ones of about 6bp in length being preferred. The oligos may be labelled withchemically-detectable or radioactive labels, with ³²P-labelled probesbeing generally preferred, and ³³P-labelled probes being even morepreferred. The kits may also comprise a chemical or other ligatingagent, such as a DNA ligase enzyme. A variety of other additionalcompositions and materials may be included in the kits, such as 96-tipor 96-pin devices, buffers, reagents for cutting long nucleic acidmolecules and tools for the size selection of DNA fragments. The kitsmay even include labelled RNA probes so that the probes may be removedby RNAase treatment and the sequencing chips re-used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Basic steps in the hybridization process. Step 1: The unlabelledtarget DNA to be sequenced (T) is hybridized under discriminativeconditions to an array of attached oligonucleotide probes. Spots withprobe Fx and Fy are depicted. Complementary sequences for Fx and Fy areat different positions of T. Step 2: Labeled probes, Pi, (one probe perchip) are hybridized to the array. Depicted is a probe that has acomplementary target on T that is adjacent to the Fx but not to the Fy.Step 3: By applying discriminative conditions or reagents, complexeswith no adjacent probes are selectively melted. A particular example isthe ligation of a labelled probe to a fixed probe, when the labelledprobe hybridizes “back to back” with the attached probe. Positivesignals are detected only in the case of adjacent probes, like Fx andPi, and in a particular example, only in the case of ligated probes.

FIG. 2A, FIG. 2B and FIG. 2C represent components of an exemplarysequencing kit.

FIG. 2A. Sequencing chips, representing an array of 4^(P) identicalsections each containing identical (or different) arrays ofoligonucleotides. Sections can be separated by physical barriers or byhydrophobic strips. 4,000-16,000 oligochips are contemplated to be inthe array.

FIG. 2B is an enlargement of a chip section containing 4^(F) spots witheach with a particular oligonucleotide probe (4,000-16,000) synthesizedor spotted on that area. Spots can be as small as several microns andthe size of the section is about 1 mm to about 10 mm.

FIG. 2C represents a set of tubes, or one or more multiwell plates, withan appropriate number of wells (in this case 4^(P) wells). Each wellcontains an amount of a specific labeled oligonucleotide. Additionalamounts of the probes can be stored unlabeled if the labeling is notdone during synthesis; in this case a sequencing kit will containnecessary components for probe labeling. The lines that are connectingtubes/wells with chip sections depict a step in the sequencing procedurewhere an amount of a labeled probe is transferred to a chip section. Thetransferring can be done by pipetting (single or multi-channel) or bypin array transferring liquid by surface tension. Transferring tools canbe also included in the sequencing kit.

FIG. 3A, FIG. 3B and FIG. 3C. Hybridization of DNA fragments produced bya random cutting of an amount of a DNA molecule. In FIG. 3A, DNAfragment T1 is such that if contains complete targets for both fixed andnon-fixed-labeled probes. FIG. 3B represents the case where the DNAfragment T is not appropriately cut. In FIG. 3C, there is enough spacefor probe P to hybridize, but the adjacent sequence is not complementaryto it. In both case B and case C, the signal will be reduced due tosaturation of the molecules of attached probe F. Simultaneoushybridization with DNA fragments and labeled probes and cycling of thehybridization process are some possible ways to increase the yield ofcorrect adjacent hybridizations.

FIG. 4. Use of Universal Base as a linker or in the terminal positionfor hybridization. The universal bases (M base, Nichols et al., 1994) orall four bases may be added in the probe synthesis. This is a way toincrease the length of the probes, and thus stability of the duplexeswithout increasing the number of probes. Also the use of universal basesat the free end of probes provides a spacer that allow the sequence tobe read in a different frame.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Determining the sequences of nucleic acid molecules is of vital use inall areas of basic and applied biological research (Drmanac &Crkvenjakov, 1990). The present invention provides new and efficientmethods for use in sequencing and analyzing nucleic acid molecules. Oneintended use for this methodology is, in conjunction with othersequencing techniques, for work on the Human Genome Project (HGP).

Presently, two methods of sequencing by hybridization, SBH, are known.In the first, Format 1, unknown genomic DNAs or oligonucleotides of upto about 100-2000 nucleotides in length are arrayed on a solidsubstrate. These DNAs are then interrogated by hybridization with a setof labeled probes which are generally 6- to 8-mers. In the inversetechnique, Format 2, oligomers of 6 to 8 nucleotides are immobilized ona solid support and allowed to anneal to pieces of cloned and labeledDNA.

In either type of SBH analysis, many steps must be included in order toarrive at a definitive sequence. Particular problems of current SBHmethods are those associated with the synthesis of large numbers ofprobes and the difficulties of effective discriminative hybridization.Full match-mismatch discrimination is difficult due to two main reasons.Firstly, the end mismatch of probes longer than 10 bases is veryundiscriminative, and secondly, the complex mixture of labeled DNAsegments that result when analyzing a long DNA fragment generates a highbackground.

The present invention provides effective discriminative hybridizationwithout large numbers of probes or probes of increased length, and alsoeliminates many of the labeling and cloning steps which are particulardisadvantages of each of the known SBH methods. The disclosed highlyefficient nucleic acid sequencing methods, termed Format 3 sequencing,are based upon hybridization with two sets of small oligonucleotideprobes of known sequences, and thus at least double the length ofsequence that can be determined. These methods allow extremely largenucleic acid molecules, including chromosomes, to be sequenced and solvevarious other SBH problems such as, for example, the attachment orlabelling of many nucleic acid fragments. The invention is extremelypowerful as it may also be used to sequence RNA and even unamplified RNAsamples.

Subsequent to the present invention, as disclosed in U.S. Ser. No.08/127,402 and in Drmanac (1994), another variation of SBH was describedtermed positional SBH (PSBH) (Broude et al., 1994). PSBH is basically avariant of Format 2 SBH (in which oligonucleotides of known sequencesare immobilized and used to hybridize to nucleic acids of unknownsequence that have been previously labelled). In PSBH, the immobilizedprobes, rather than being simple, single-stranded probes, are duplexesthat contain single stranded 3′ overhangs. Biotinylated duplex probesare immobilized on streptavidin-coated magnetic beads, to form a type ofimmobilized probe, and then mixed with ³²P-labeled target nucleic acidsto be sequenced. T4 DNA ligase is then added to ligate any hybridizedtarget DNA to the shorter end of the duplex probe.

However, despite representing an interesting approach, PSBH (as reportedby Broude et al., 1994) does not reflect a significant advance over theexisting SBH technology. For example, unlike the Format 3 methodology ofthe present invention, PSBH does not extend the length of sequence thatcan be determined in one round of the method. PSBH also maintains theburdensome requirement for labelling the unknown target DNA, which isnot required for Format 3. In general, PSBH is proposed for use incomparative studies or in mapping, rather than in de novo genomesequencing. It thus differs significantly from Format 3 which, althoughwidely applicable to all areas of sequencing, is a very powerful toolfor use in sequencing even the largest of genomes.

The nucleic acids to be sequenced may first be fragmented. This may beachieved by any means including, for example, cutting by restrictionenzyme digestion, particularly with Cvi JI as described by Fitzgerald etal. (1992); shearing by physical means such as ultrasound treatment; byNaOH treatment, and the like. If desired, fragments of an appropriatelength, such as between about 10 bp and about 40 bp may be cut out of agel. The complete nucleic acid sequence of the original molecule, suchas a human chromosome, would be determined by defining F+P sequencespresent in the original molecule and assembling portions of overlappingF+P sequences. This does not, therefore, require an intermediate step ofdetermining fragment sequences, rather, the sequence of the wholemolecule is constructed from F+P sequences delineated.

For the purposes of the following discussion, it will be generallyassumed that four bases make up the sequences of the nucleic acids to besequenced. These are A, G, C and T for DNA and A, G, C and U for RNA.However, it may be advantageous in certain embodiments to use modifiedbases in the small oligonucleotide probes. To carry out the invention,one would generally first prepare a number of small oligonucleotideprobes of defined length that cover all combinations of sequences forthat length of probe. This number is represented by 4^(N) (4 to thepower N) where the length of the probe is termed N. For example, thereare 4096 possible sequences for a 6-mer probe (4⁶=4096).

One set of such probes of length F (4^(F)) would be fixed in a squarearray on a microchip—which may be in the range of 1 mm² or 1 cm². In thepresent example, these would be arranged in 64 rows and 64 columns.Naturally, one would ensure that the oligo probes were attached, orotherwise immobilized, to the microchip surface so that were able totake part in hybridization reactions. Another set of oligos of length P,4^(P) in number, would be also synthesized. The oligos in this “P set”would be labeled with a detectable label and would be dispensed into aset of tubes (FIG. 2A, FIG. 2B and FIG. 2C).

4^(P) of the chips would be combined in a large array (or several arraysof approximately 10-100 cm², for a convenient size); where P correspondsto the length of oligonucleotides in the second oligomer set (FIG. 2A,FIG. 2B and FIG. 2C). Again, as a convenient example, P is chosen to besix (P=6).

The nucleic acids to be sequenced would be fragmented to give smallernucleic acid fragments of unknown sequence. The average length of thesefragments, termed T. should generally be greater than the combinedlength of F and P and may be about three times the length of F (i.e.,F+P≦T and T≈3F). In the present example, one would aim to producenucleic acid fragments of approximately 20 base pairs in length. Thesefragments would be denatured and added to the large arrays underconditions that facilitate hybridization of complementary sequences.

In the simplest and currently preferred form of the invention,hybridization conditions would be chosen that would allow significanthybridization to occur only if 6 sequential nucleotides in a nucleicacid fragment were complementary to all 6 nucleotides of an Foligonucleotide probe. Such hybridization conditions would be determinedby routine optimization pilot studies in which conditions such as thetemperature, the concentration of various components, the length of timeof the steps, and the buffers used, including the pH of the buffer.

At this stage, each microchip would contain certain hybridizedcomplexes. These would be in the form of probe:fragment complexes inwhich the entire sequence of the probe is hybridized to the fragment,but in which the fragment, being longer, has some non-hybridizedsequences that form a “tail” or “tails” to the complex. In this example,the complementary hybridized sequences would be of length F and thenon-hybridized sequences would total T−F in length. The complementaryportion of the fragment may be at or towards an appropriate end, so thata single longer non-hybridized tail is formed. Alternatively, thecomplementary portion of the fragment may be towards the opposite end,so that two non-hybridized tails are formed (FIG. 3A, FIG. 3B and FIG.3C).

After washing to remove the non-complementary nucleic acid fragmentsthat did not hybridize, a small amount of the labeled oligonucleotidesin set P would be added to each microchip for hybridization to thenucleic acid fragment tails of unknown sequence that protrude from theprobe:fragment complexes. Only one of each of the 4^(P) nucleotideswould be added to each microchip. Again, it is currently preferred touse hybridization conditions that would allow significant binding tooccur only if all the 6 nucleotides of a labelled probe werecomplementary to 6 sequential nucleotides of a nucleic acid fragmenttail. The hybridization conditions would be determined by pilot studies,as described above, in which components such as the temperature,concentration, time, buffers and the like, are optimized.

At this stage, each microchip would then contain certain ‘secondaryhybridized complexes’. These would be in the form ofprobe:fragment:probe complexes in which the entire sequence of eachprobe is hybridized to the fragment, and in which the fragment likelyhas some non-hybridized sequences. In these secondary hybridizedcomplexes the immobilized probe and the labelled probe may be hybridizedto the fragment so that the two probes are immediately adjacent or “backto back”. However, given that the fragments will generally be longerthan the sum of the lengths of the probes, the immobilized probe and thelabelled probe may be hybridized to the fragment in non-adjacentpositions separated by one or more bases.

The large arrays would then be treated by a process to remove thenon-hybridized labelled probes. In preferred embodiments, the processemployed would remove not only the non-hybridized labelled probes, butalso the non-adjacently-hybridized labelled probes from the array. Theprocess would employ discriminating conditions to allow those secondaryhybridization complexes that include adjacent immobilized and labelledprobes to be discriminating from those secondary hybridization complexesin which the nucleic acid fragment is hybridized to two probes but whichprobes are not adjacent. This is an important aspect of the invention inthat it will allow the ultimate delineation of a section of fragmentsequence corresponding to the combined sequences of the immobilizedprobe and the labelled probe.

The discrimination process employed to remove non-hybridized andnon-adjacently-hybridized probes from the array whilst leaving theadjacently-hybridized probes attached may again be a controlled washingprocess. The adjacently-hybridized probes would be unaffected by thechosen conditions by virtue of their increased stability due to thestacking reactions of the adjacent nucleotides. However, in preferredembodiments, it is contemplated that one would treat the large arrays sothat any adjacent probes would be covalently joined, e.g., by treatingwith a solution containing a chemical ligating agent or, morepreferably, a ligase enzyme, such as T₄ DNA ligase (Landegren et al.1988; Wu & Wallace, 1989).

In any event, the complete array would be subjected to stringent washingso that the only label left associated with the array would be in theform of double-stranded probe-fragment-probe complexes with adjacenthybridized portions of length F+P (i.e., 12 nucleotides in the presentexample). Using this two step hybridization reaction, very highdiscrimination is possible because three or four independentdiscriminative processes are taken into account: discriminativehybridization of fragment T to F bases long probe; discriminativehybridization of P bases long probe to fragment T; discriminativestability of full match (F+T+P) hybrid in comparison to P hybrids oreven to mismatched hybrids containing non-adjacent F+P probes; anddiscriminative ligation of the two end bases of F and P.

One would then detect the so-called adjacent secondary complexes byobserving the location of the remaining label on the array. From theposition of the label, F+P (e.g., 12) nucleotide long sequences from thefragment could be determined by combining the known sequences of theimmobilized and labelled probes. The complete nucleic acid sequence ofthe original molecule, such as a human chromosome, could then bereconstructed or assembled from the overlapping F+P sequences thusdetermined.

When ligation is employed in the sequencing process, as is currentlypreferred, then the ordinary oligonucleotides chip cannot be reused. Theinventor contemplates that this will not be limiting as various methodsare available for recycling. For example, one may generate aspecifically cleavable bond between the probes and then cleave the bondafter detection. Alternatively, one may employ ribonucleotides for thesecond probe, probe P, or use a ribonucleotide for the joining base inprobe P, so that this probe may subsequently be removed by RNAase oruracil-DNA glycosylate treatment (Craig et al., 1989). Othercontemplated methods are to establish bonds by chemical ligation whichcan be selectively cut (Dolinnaya et al., 1988).

Further variations and improvements to this sequencing methodology arealso contemplated and fall within the scope of the present invention.This includes the use of modified oligonucleotides to increase thespecificity or efficiency of the methods, similar to that described byHoheisel & Lehrach (1990). Cycling hybridizations can also be employedto increase the hybridization signal, as is used in PCR technology. Inthese cases, one would use cycles with different temperatures tore-hybridize certain probes. The invention also provides for determiningshifts in reading frames by using equimolar amounts of probes that havea different base at the end position. For example, using equimolar7-mers in which the first six bases are the same defined sequence andthe last position may be A, T, C or G in the alternative.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE I Preparation of Support Bound Oligonucleotides

Oligonucleotides, i.e., small nucleic acid segments, may be readilyprepared by, for example, directly synthesizing the oligonucleotide bychemical means, as is commonly practiced using an automatedoligonucleotide synthesizer.

Support bound oligonucleotides may be prepared by any of the methodsknown to those of skill in the art using any suitable support such asglass, polystyrene or teflon. One strategy is to precisely spotoligonucleotides synthesized by standard synthesizers. Immobilizationcan be achieved using passive adsorption (Inouye & Hondo, 1990); usingUV light (Nagata et al., 1985; Dahlen et al., 1987; Morriey & Collins,1989) or by covalent binding of base modified DNA (Keller et al., 1988;1989); all references being specifically incorporated herein.

Another strategy that may be employed is the use of the strongbiotin-streptavidin interaction as a linker. For example, Broude et al.(1994) describe the use of biotinylated probes, although these areduplex probes, that are immobilized on streptavidin-coated magneticbeads. Streptavidin-coated beads may be purchased from Dynal, Oslo. Ofcourse, this same linking chemistry is applicable to coating any surfacewith streptavidin. Biotinylated probes may be purchased from varioussources, such as, e.g., Operon Technologies (Alameda, Calif.).

Nunc Laboratories (Naperville, Ill.) is also selling suitable materialthat could be used. Nunc Laboratories have developed a method by whichDNA can be covalently bound to the microwell surface termed CovaLink NH.CovaLink NH is a polystyrene surface grafted with secondary amino groups(>NH) that serve as bridge-heads for further covalent coupling. CovaLinkModules may be purchased from Nunc Laboratories. DNA molecules may bebound to CovaLink exclusively at the 5′-end by a phosphoramidate bond,allowing immobilization of more than 1 pmol of DNA (Rasmussen et al.,1991).

The use of CovaLink NH strips for covalent binding of DNA molecules atthe 5′-end has been described (Rasmussen et al., 1991). In thistechnology, a phosphoramidate bond is employed (Chu et al., 1983). Thisis beneficial as immobilization using only a single covalent bond ispreferred. The phosphoramidate bond joins the DNA to the CovaLink NHsecondary amino groups that are positioned at the end of spacer armscovalently grafted onto the polystyrene surface through a 2 nm longspacer arm. To link an oligonucleotide to CovaLink NH via anphosphoramidate bond, the oligonucleotide terminus must have a 5′-endphosphate group. It is, perhaps, even possible for biotin to becovalently bound to CovaLink and then streptavidin used to bind theprobes.

More specifically, the linkage method includes dissolving DNA in water(7.5 ng/μl) and denaturing for 10 min. at 95° C. and cooling on ice for10 min. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm₇), is thenadded to a final concentration of 10 mM 1-MeIm₇. A ss DNA solution isthen dispensed into CovaLink NH strips (75 μl/well) standing on ice.

Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC),dissolved in 10 mM 1-MeIm₇, is made fresh and 25 μl added per well. Thestrips are incubated for 5 hours at 50° C. After incubation the stripsare washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3times, then they are soaked with washing solution for 5 min., andfinally they are washed 3 times (wherein he washing solution is 0.4 NNaOH, 0.25% SDS heated to 50° C.).

It is contemplated that a further suitable method for use with thepresent invention is that described in PCT Patent Application WO90/03382 (Southern & Maskos), incorporated herein by reference. Thismethod of preparing an oligonucleotide bound to a support involvesattaching a nucleoside 3′-reagent through the phosphate group by acovalent phosphodiester link to aliphatic hydroxyl groups carried by thesupport. The oligonucleotide is then synthesized on the supportednucleoside and protecting groups removed from the syntheticoligonucleotide chain under standard conditions that do not cleave theoligonucleotide from the support. Suitable reagents include nucleosidephosphoramidite and nucleoside hydrogen phosphorate.

In more detail, to use this method, a support, such as a glass plate, isderivatized by contact with a mixture of xylene,glycidoxypropyltrimethoxysilane, and a trace of diisopropylethylamine at90° C. overnight. It is then washed thoroughly with methanol, ether andair-dried. The derivatized support is then heated with stirring inhexaethyleneglycol containing a catalytic amount of concentratedsulfuric acid, overnight in an atmosphere of argon, at 80° C., to yieldan alkyl hydroxyl derivatized support. After washing with methanol andether, the support is dried under vacuum and stored under argon at −20°C.

Oligonucleotide synthesis is then performed by hand under standardconditions using the derivatized glass plate as a solid support. Thefirst nucleotide will be a 3′-hydrogen phosphate, used in the form ofthe triethylammonium salt. This method results in support boundoligonucleotides of high purity.

An on-chip strategy for the preparation of DNA probe arrays may beemployed. For example, addressable laser-activated photodeprotection maybe employed in the chemical synthesis of oligonucleotides directly on aglass surface, as described by Fodor et al. (1991), incorporated hereinby reference. Probes may also be immobilized on nylon supports asdescribed by Van Ness et al. (1991); or linked to teflon using themethod of Duncan & Cavalier (1988); all references being specificallyincorporated herein.

Fodor et al. (1991) describe the light-directed synthesis ofdinucleotides which is applicable to the spatially directed synthesis ofcomplex compounds for use in the microfabrication of devices. This isbased upon a method that uses light to direct the simultaneous synthesisof chemical compounds on a solid support. The pattern of exposure tolight or other forms of energy through a mask, or by other spatiallyaddressable means, determines which regions of the support are activatedfor chemical coupling. Activation by light results from the removal ofphotolabile protecting groups from selected areas. After deprotection, afirst compound bearing a photolabile protecting group is exposed to theentire surface, but reaction occurs only with regions that wereaddressed by light in the preceding step. The substrate is thenilluminated through a second mask, which activates a different regionfor reaction with a second protected building block. The pattern ofmasks used in these illuminations and the sequence of reactants definethe ultimate products and their locations. A high degree ofminiaturization is possible with the Fodor method because the density ofsynthesis sites is bounded only by physical limitations on spatialaddressability, i.e., the diffraction of light. Each compound isaccessible and its position is precisely known. hence, an oligo chipmade in this way would be ready for use in SBH.

Fodor et al. (1991) describes the light-activated formation of adinucleotide as follows. 5′-Nitroveratryl thymidine was synthesized fromthe 3′-O-thymidine acetate. After deprotection with base, the5′-nitroveratryl thymidine was attached to an aminated substrate througha linkage to the 3′-hydroxyl group. The nitrovertryl protecting groupswere removed by illumination through a 500-μm checkerboard mask. Thesubstrate was then treated with phosphoramidite-activated2′-deoxycytidine. In order to follow the reaction fluorometrically, thedeoxycytidine had been modified with an FMOC-protected aminohexyl linkerattached to the exocyclic amine. After removal of the FMOC protectinggroup with base, the regions that contained the dinucleotide werefluorescently labeled by treatment of the substrate with FITC.Therefore, following this method, support bound-oligonucleotides can besynthesized.

To link an oligonucleotide to a nylon support, as described by Van Nesset al. (1991), requires activation of the nylon surface via alkylationand selective activation of the 5′-amine of oligonucleotides withcyanuric chloride, as follows. A nylon surface is ethylated usingtriethyloxonium tetrafluoroborate to form amine reactive imidate esterson the surface of the nylon 1-methyl-2-pyrrolidone is used as a solvent.The nylon surface is unpolished to effect the greatest possible surfacearea.

The activated surface is then reacted with poly(ethyleneimine)(M_(r)˜10K-70K) to form a polymer coating that provides an extendedamine surface for the attachment of oligos. Amine-tailedoligonucleotide(s) selectively react with excess cyanuric chloride,exclusively on the amine tail, to give a4,6-dichloro-1,3,5-triazinyl-oligonucleotide(s) in quantitative yield.The displacement of one chlorine moiety of cyanuric chloride by theamino group significantly diminishes the reactivity of the remainingchlorine groups. This results in increased hydrolytic stability of the4,6-dichloro-1,3,5-triazinyl-oligonucleotide(s) are stable for extendedperiods in buffered aqueous solutions (pH 8.3, 4° C., 1 week) and arereadily isolated and purified by size elusion chromatography orultrafiltration.

The reaction is specific for the amine tail with no apparent reaction onthe nucleotide moieties. The PEI-coated nylon surface is then reactedwith the cyanuric chloride activated oligonucleotide. Highconcentrations of the ‘capture’ sequence are readily immobilized on thesurface and the unreacted amines are capped with succinic anhydride inthe final step of the derivatization process.

One particular way to prepare support bound oligonucleotides is toutilize the light-generated synthesis described by Pease et al. (1994,incorporated herein by reference). These authors used currentphotolithographic techniques to generate arrays of immobilizedoligonucleotide probes (DNA chips). These methods, in which light isused to direct the synthesis of oligonucleotide probes in high-density,miniaturized arrays, utilize photolabile 5′-protectedN-acyl-deoxynucleoside phosphoramidites, surface linker chemistry andversatile combinatorial synthesis strategies. A matrix of 256 spatiallydefined oligonucleotide probes may be generated in this manner and thenused in the advantageous Format 3 sequencing, as described herein.

Pease et al. (1994) presented a strategy suitable for use inlight-directed oligonucleotide synthesis. In this method, the surface ofa solid support modified with photolabile protecting groups isilluminated through a photolithographic mask, yielding reactive hydroxylgroups in the illuminated regions. A 3′O-phosphoramidite-activateddeoxynucleoside (protected at the 5′-hydroxyl with a photolabile group)is then presented to the surface and coupling occurs at sites that wereexposed to light. Following capping, and oxidation, the substrate isrinsed and the surface is illuminated through a second mask, to exposeadditional hydroxyl groups for coupling. A second 5′-protected, 3′O-phosphoramidite-activated deoxynucleoside is presented to the surface.The selective photodeprotection and coupling cycles are repeated untilthe desired set of products is obtained. Since photolithography is used,the process can be miniaturized to generate high-density arrays ofoligonucleotide probes, the sequence of which is known at each site.

The synthetic pathway for preparing the necessary5′O-(α-methyl-6-nitropiperonyloxycabonyl)-N-acyl-2′-deoxynucleosidephosphoramidites (MeNPoc-N-acyl-2′-deoxynucleoside phophoramidites)involves, in the first step, an N-acyl-2′-deoxynucleoside that reactswith 1-(2-nitro-4,5-methylenedioxyphenyl)ethyl-1-chloroformate to yield5′-MeNPoc-N-acyl-2′-deoxynucleoside. In the second step, the 3′-hydroxylreacts with 2-cyanoethyl N,N′-diisopropylchlorophosphoramidite, usingstandard procedures, to yield the5′-MeNPoc-N-acyl-2′-deoxynucleoside-3′-O-(2-cyanoethyl-N-N-diisopropyl)phosphoramidites.The photoprotecting group is stable under ordinary phosporamiditesynthesis conditions and can be removed with aqueous base. Thesereagents can be stored for long periods under argon at 4° C.

Photolysis half-times of 28 s, 31 s, 27 s, and 18 s for MeNPoc-dT,MeNPoc-dC^(ib), MeNPoc-dG^(PAC), and MeNPoc-dA^(PAC) respectively, havebeen reported (Pease et al., 1994). In lithographic synthesis,illumination times of 4.5 min (9×t_(1/2)MeNPoc-dC) are thereforerecommended to ensure >99% removal of MeNPoc protecting groups.

A suitable synthetic support is one consisting of a 5.1×7.6 cm glasssubstrate prepared by cleaning in concentrated NaOH, followed byexhaustive rinsing in water. The surfaces would then be derivatized for2 hr with a solution of 10% (vol/vol)bis(2-hydroxyethyl)aminopropyltriethoxysilane (Petrarch Chemicals,Bristol, Pa.) in 95% ethanol, rinsed thoroughly with ethanol and ether,dried in vacuo at 40° C., and heated at 100° C. for 15 min. In suchstudies, a synthesis linker would be attached by reacting derivatizedsubstrates with 4,4′-dimethoxytrityl (DMT)-hexaethyloxy-O-cyanoethylphosphoramidite.

In summary, to initiate the synthesis of an oligonucleotide probe, theappropriate deoxynucleoside phosphoramidite derivative would be attachedto a synthetic support through a linker. Regions of the support are thenactivated for synthesis by illumination through, e.g., 800×12800 μmapertures of a photolithographic mask. Additional phosphoramiditesynthesis cycles may be performed (with DMT-protected deoxynucleosides)to generate any required sequence, such as any 4-,5-,6-,7-,8-,9- or even10-mer sequence. Following removal of the phosphate and exocyclic amineprotecting groups with concentrated NH₄OH for 4 hr, the substrate maythen be mounted in a water-jacketed thermostatically controlledhybridization chamber, ready for use.

Of course, one could easily purchase a DNA chip, such as one of thelight-activated chips described above, from a commercial source. In thisregard, one may contact Affymetrix of Santa Clara, Calif. 95051, andBeckman.

EXAMPLE II Modified Oligonucleotides for Use in Probes

Modified oligonucleotides may be used throughout the procedures of thepresent invention to increase the specificity or efficiency ofhybridization. A way to achieve this is the substitution of naturalnucleotides by base modification. For example, pyrimidines with ahalogen at the C⁵-position may be used. This is believed to improveduplex stability by influencing base stacking. 2,6-diaminopurine mayalso be used to give a third hydrogen bond in its base pairing withthymine, thereby thermally stabilizing DNA-duplexes. Using2,6-diaminopurine is reported to lead to a considerable improvement inthe duplex stability of short oligomers. Its incorporation is proposedto allow more stringent conditions for primer annealing, therebyimproving the specificity of the duplex formation and suppressingbackground problems or the use of shorter oligomers.

The synthesis of the triphosphate versions of these modified nucleotidesis disclosed by Hoheisel & Lehrach (1990, incorporated herein byreference). Briefly, 5-Chloro-2′-deoxyuridine and 2,6-diaminopurine2′-deoxynucleoside are purchased, e.g., from Sigma. Phosphorylation iscarried out as follows: 50 mg dry 2-NH₂-dAdo is taken up in 500 μl drytriethyl phosphate stirring under argon. 25 μl POCl₃ is added and themixture incubated at −20° C. In the meantime, 1 mmol pyrophosphoric acidis dissolved in 0.95 ml tri-n-butylamine and 2 ml methanol and dried ina rotary evaporator. Subsequently it is dried by evaporation twice from5 ml pyridine, with 70 μl tri-n-butylamine also added before the secondtime. Finally it is dissolved in 2 ml dry dimethyl formamide.

After 90 min at −20° C., the phosphorylation mixture is evaporated toremove excess POCl₃ and the tri-n-butylammonium pyrophosphate indimethyl formamide is added. Incubation is for 1.5 min at roomtemperature. The reaction is stopped by addition of 5 ml 0.2 Mtriethylammonium bicarbonate (pH 7.6) and kept on ice for 4 hours. For5-C1-dUrd, the conditions would be identical, but 50 μl POCl₃ would beadded and the phosphorylation carried out at room temperature for 4hours.

After the hydrolysis, the mixture is evaporated, the pH adjusted to 7.5,and extracted with 1 volume diethyl ether. Separation of the productsis, e.g., on a (2.5×20 cm) Q-Sepharose column using a linear gradient of0.15 M to 0.8 M triethylammonium bicarbonate. Stored frozen, thenucleotides are stable over long periods of time.

One may also use the non-discriminatory base analogue, or universalbase, as designed by Nichols et al. (1994). This new analogue,1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole (designated M), wasgenerated for use in oligonucleotide probes and primers for solving thedesign problems that arise as a result of the degeneracy of the geneticcode, or when only fragmentary peptide sequence data are available. Thisanalogue maximizes stacking while minimizing hydrogen-bondinginteractions without sterically disrupting a DNA duplex.

The M nucleoside analogue was designed to maximize stacking interactionsusing aprotic polar substituents linked to heteroaromatic rings,enhancing intra- and inter-strand stacking interactions to lessen therole of hydrogen bonding in base-pairing specificity. Nichols et al.(1994) favored 3-nitropyrrole 2′-deoxyribonucleoside because of itsstructural and electronic resemblance to p-nitroaniline, whosederivatives are among the smallest known intercalators ofdouble-stranded DNA.

The dimethoxytrityl-protected phosphoramidite of nucleoside M is alsoavailable for incorporation into nucleotides used as primers forsequencing and polymerase chain reaction (PCR). Nichols et al. (1994)showed that a substantial number of nucleotides can be replaced by Mwithout loss of primer specificity.

A unique property of M is its ability to replace long strings ofcontiguous nucleosides and still yield functional sequencing primers.Sequences with three, six and nine M substitutions have all beenreported to give readable sequencing ladders, and PCR with threedifferent M-containing primers all resulted in amplification of thecorrect product (Nichols et al., 1994).

The ability of 3-nitropyrrole-containing oligonucleotides to function asprimers strongly suggests that a duplex structure must form withcomplementary strands. Optical thermal profiles obtained for theoligonucleotide pairs d(5′-C₂-T₅XT₅G₂-3′) and d(5′-C₂A₅YA₅G₂-3′) (whereX and Y can be A, C, G, T or M) were reported to fit the normalsigmoidal pattern observed for the DNA double-to-single strandtransition. The T_(m) values of the oligonucleotides containing X.M basepairs (where X was A, C, G or T, and Y was M) were reported to all fallwithin a 3° C. range (Nichols et al., 1994).

EXAMPLE III Preparation of Sequencing Chips and Arrays

The present example describes physical embodiments of sequencing chipscontemplated by the inventor.

A basic example is using 6-mers attached to 50 micron surfaces to give achip with dimensions of 3×3 mm which can be combined to give an array of20×20 cm. Another example is using 9-mer oligonucleotides attached to10×10 microns surface to create a 9-mer chip, with dimensions of 5×5 mm.4000 units of such chips may be used to create a 30×30 cm array. FIG.2A, FIG. 2B and FIG. 2C illustrate yet another example of an array inwhich 4,000 to 16,000 oligochips are arranged into a square array. Aplate, or collection of tubes, as also depicted, may be packaged withthe array as part of the sequencing kit.

The arrays may be separated physically from each other or by hydrophobicsurfaces. One possible way to utilize the hydrophobic strip separationis to use technology such as the Iso-Grid Microbiology System producedby QA Laboratories, Toronto, Canada.

Hydrophobic grid membrane filters (HGMF) have been in use in analyticalfood microbiology for about a decade where they exhibit uniqueattractions of extended numerical range and automated counting ofcolonies. One commercially-available grid is ISO-GRID™ from QALaboratories Ltd. (Toronto, Canada) which consists of a square (60×60cm) of polysulfone polymer (Gelman Tuffryn HT-450, 0.45μ pore size) onwhich is printed a black hydrophobic ink grid consisting of 1600 (40×40)square cells. HGMF have previously been inoculated with bacterialsuspensions by vacuum filtration and incubated on the differential orselective media of choice.

Because the microbial growth is confined to grid cells of known positionand size on the membrane, the HGMF functions more like an MPN apparatusthan a conventional plate or membrane filter. Peterkin et al. (1987)reported that these HGMFs can be used to propagate and store genomiclibraries when used with a HGMF replicator. One such instrumentreplicates growth from each of the 1600 cells of the ISO-GRID andenables many copies of the master HGMF to be made (Peterkin et al.,1987).

Sharpe et al. (1989) also used ISO-GRID HGMF from QA Laboratories and anautomated HGMF counter (MI-100 Interpreter) and RP-100 Replicator. Theyreported a technique for maintaining and screening many microbialcultures.

Peterkin and colleagues later described a method for screening DNAprobes using the hydrophobic grid-membrane filter (Peterkin et al.,1989). These authors reported methods for effective colony hybridizationdirectly on HGMFs. Previously, poor results had been obtained due to thelow DNA binding capacity of the polysulfone polymer on which the HGMFsare printed. However, Peterkin et al. (1989) reported that the bindingof DNA to the surface of the membrane was improved by treating thereplicated and incubated HGMF with polyethyleneimine, a polycation,prior to contact with DNA. Although this early work uses cellular DNAattachment, and has a different objective to the present invention, themethodology described may be readily adapted for format 3 SBH.

In order to identify useful sequences rapidly, Peterkin et al. (1989)used radiolabeled plasmid DNA from various clones and tested itsspecificity against the DNA on the prepared HGMFs. In this way, DNA fromrecombinant plasmids was rapidly screened by colony hybridizationagainst 100 organisms on HGMF replicates which can be easily andreproducibly prepared.

Two basic problems have to be solved. Manipulation with small (2-3 mm)chips, and parallel execution of thousands of the reactions. Thesolution of the invention is to keep the chips and the probes in thecorresponding arrays. In one example, chips containing 250,000 9-mersare synthesized on a silicon wafer in the form of 8×8 mM plates (15μM/oligonucleotide, Pease et al., 1994) arrayed in 8×12 format (96chips) with a 1 mM groove in between. Probes are added either bymultichannel pipet or pin array, one probe on one chip. To score all4000 6-mers, 42 chip arrays have to be used, either using differentones, or by reusing one set of chip arrays several times.

In the above case, using the earlier nomenclature of the application,F=9; P=6; and F+P=15. Chips may have probes of formula BxNn, where x isa number of specified bases B; and n is a number of non-specified bases,so that x=4 to 10 and n=1 to 4. To achieve more efficient hybridization,and to avoid potential influence of any support oligonucleotides, thespecified bases can be surrounded by unspecified bases, thus representedby a formula such as (N)nBx(N)m (FIG. 4).

EXAMPLE IV Preparation of Nucleic Acid Fragments

The nucleic acids to be sequenced may be obtained from any appropriatesource, such as cDNAs, genomic DNA, chromosomal DNA, microdissectedchromosome bands, cosmid or YAC inserts, and RNA, including mRNA withoutany amplification steps. For example, Sambrook et al. (1989) describesthree protocols for the isolation of high molecular weight DNA frommammalian cells (p. 9.14-9.23).

The nucleic acids would then be fragmented by any of the methods knownto those of skill in the art including, for example, using restrictionenzymes as described at 9.24-9.28 of Sambrook et al. (1989), shearing byultrasound and NaOH treatment.

Low pressure shearing is also appropriate, as described by Schriefer etal. (1990, incorporated herein by reference). In this method, DNAsamples are passed through a small French pressure cell at a variety oflow to intermediate pressures. A lever device allows controlledapplication of low to intermediate pressures to the cell. The results ofthese studies indicate that low-pressure shearing is a usefulalternative to sonic and enzymatic DNA fragmentation methods.

One particularly suitable way for fragmenting DNA is contemplated to bethat using the two base recognition endonuclease, CviJI, described byFitzgerald et al. (1992). These authors described an approach for therapid fragmentation and fractionation of DNA into particular sizes thatthey contemplated to be suitable for shotgun cloning and sequencing. Thepresent inventor envisions that this will also be particularly usefulfor generating random, but relatively small, fragments of DNA for use inthe present sequencing technology.

The restriction endonuclease CviJI normally cleaves the recognitionsequence PuGCPy between the G and C to leave blunt ends. Atypicalreaction conditions, which alter the specificity of this enzyme(CviJI**), yield a quasi-random distribution of DNA fragments from thesmall molecule pUC19 (2688 base pairs). Fitzgerald et al. (1992)quantitatively evaluated the randomness of this fragmentation strategy,using a CviJI** digest of pUC19 that was size fractionated by a rapidgel filtration method and directly ligated, without end repair, to alacZ minus M13 cloning vector. Sequence analysis of 76 clones showedthat CviJI** restricts PyGCPy and PuGCPu, in addition to PuGCPy sites,and that new sequence data is accumulated at a rate consistent withrandom fragmentation.

As reported in the literature, advantages of this approach compared tosonication and agarose gel fractionation include: smaller amounts of DNAare required (0.2-0.5 μg instead of 2-5 μg); and fewer steps areinvolved (no preligation, end repair, chemical extraction, or agarosegel electrophoresis and elution are needed). These advantages are alsoproposed to be of use when preparing DNA for sequencing by Format 3.

Irrespective of the manner in which the nucleic acid fragments areobtained or prepared, it is important to denature the DNA to give singlestranded pieces available for hybridization. This is achieved byincubating the DNA solution for 2-5 minutes at 80-90° C. The solution isthen cooled quickly to 2° C. to prevent renaturation of the DNAfragments before they are contacted with the chip. Phosphate groups mustalso be removed from genomic DNA, as described in Example VI.

EXAMPLE V Preparation of Labelled Probes

The oligonucleotide probes may be prepared by automated synthesis, whichis routine to those of skill in the art, for example, using an AppliedBiosystems system. Alternatively, probes may be prepared using GenosysBiotechnologies Inc. methods using stacks of porous Teflon wafers.

Oligonucleotide probes may be labelled with, for example, radioactivelabels (³⁵S, ³²P, ³³P, and preferably, ³³P) for arrays with 100-200 μmor 100-400 μm spots; non-radioactive isotopes (Jacobsen et al., 1990);or fluorophores (Brumbaugh et al., 1988). All such labelling methods areroutine in the art, as exemplified by the relevant sections in Sambrooket al. (1989) and by further references such as Schubert et al. (1990),Murakami et al. (1991) and Cate et al. (1991), all articles beingspecifically incorporated herein by reference.

In regard to radiolabeling, the common methods are end-labelling usingT4 polynucleotide kinase or high specific activity labelling usingKlenow or even T7 polymerase. These are described as follows.

Synthetic oligonucleotides are synthesized without a phosphate group attheir 5′ termini and are therefore easily labeled by transfer of theγ-³²P or γ-³³P from [γ-³²P]ATP or [γ-³³P]ATP using the enzymebacteriophage T4 polynucleotide kinase. If the reaction is carried outefficiently, the specific activity of such probes can be as high as thespecific activity of the [γ-³²P]ATP or [γ-³³P]ATP itself. The reactiondescribed below is designed to label 10 pmoles of an oligonucleotide tohigh specific activity. Labeling of different amounts of oligonucleotidecan easily be achieved by increasing or decreasing the size of thereaction, keeping the concentrations of all components constant.

A reaction mixture would be created using 1.0 μl of oligonucleotide (10pmoles/μl); 2.0 μl of 10×bacteriophage T4 polynucleotide kinase buffer;5.0 μl of [γ-³²P]ATP or [γ-³³P]ATP (sp. act. 5000 Ci/mmole; 10 mCi/ml inaqueous solution) (10 pmoles); and 11.4 μl of water. Eight (8) units (˜1μl) of bacteriophage T4 polynucleotide kinase is added to the reactionmixture mixed well, and incubated for 45 minutes at 37° C. The reactionis heated for 10 minutes at 68° C. to inactivate the bacteriophage T4polynucleotide kinase.

The efficiency of transfer of ³²P or ³³P to the oligonucleotide and itsspecific activity is then determined. If the specific activity of theprobe is acceptable, it is purified. If the specific activity is toolow, an additional 8 units of enzyme is added and incubated for afurther 30 minutes at 37° C. before heating the reaction for 10 minutesat 68° C. to inactivate the enzyme.

Purification of radiolabeled oligonucleotides can be achieved byprecipitation with ethanol; precipitation with cetylpyridinium bromide;by chromatography through bio-gel P-60; or by chromatography on aSep-Pak C₁₈ column.

Probes of higher specific activities can be obtained using the Klenowfragment of E. coli. DNA polymerase I to synthesize a strand of DNAcomplementary to the synthetic oligonucleotide. A short primer ishybridized to an oligonucleotide template whose sequence is thecomplement of the desired radiolabeled probe. The primer is thenextended using the Klenow fragment of E. coli DNA polymerase I toincorporate [α-³²P]dNTPs or [α-³³P]dNTPs in a template-directed manner.After the reaction, the template and product are separated bydenaturation followed by electrophoresis through a polyacrylamide gelunder denaturing conditions. With this method, it is possible togenerate oligonucleotide probes that contain several radioactive atomsper molecule of oligonucleotide, if desired.

To use this method, one would mix in a microfuge tube the calculatedamounts of [α-³²P]dNTPs or [α-³³P]dNTPs necessary to achieve the desiredspecific activity and sufficient to allow complete synthesis of alltemplate strands. The concentration of dNTPs should not be less than 1μM at any stage during the reaction. Then add to the tube theappropriate amounts of primer and template DNAs, with the primer beingin three- to tenfold molar excess over the template.

0.1 volume of 10×Klenow buffer would then be added and mixed well. 2-4units of the Klenow fragment of E. coli DNA polymerase I would then beadded per 5 μl of reaction volume, mixed and incubated for 2-3 hours at4° C. If desired, the progress of the reaction may be monitored byremoving small (0.1-μl) aliquots and measuring the proportion ofradioactivity that has become precipitable with 10% trichloroacetic acid(TCA).

The reaction would be diluted with an equal volume of gel-loadingbuffer, heated to 80° C. for 3 minutes, and then the entire sampleloaded on a denaturing polyacrylamide gel. Following electrophoresis,the gel is autoradiographed, allowing the probe to be localized andremoved from the gel. Various methods for fluorophobic labelling arealso available, as follows. Brumbaugh et al. (1988) describe thesynthesis of fluorescently labeled primers. A deoxyuridine analog with aprimary amine “linker arm” of 12 atoms attached at C-5 is synthesized.Synthesis of the analog consists of derivatizing 2′-deoxyuridine throughorganometallic intermediates to give 5′(methylpropenoyl)-2′-deoxyuridine. Reaction with dimethoxytrityl-chlorideproduces the corresponding 5′-dimethoxytrityl adduct. The methyl esteris hydrolyzed, activated, and reacted with an appropriately monoacylatedalkyl diamine. After purification, the resultant linker arm nucleosidesare converted to nucleoside analogs suitable for chemicaloligonucleotide synthesis.

Oligonucleotides would then be made that include one or two linker armbases by using modified phosphoridite chemistry. To a solution of 50nmol of the linker arm oligonucleotide in 25 μl of 500 mM sodiumbicarbonate (pH 9.4) is added 20 μl of 300 mM FITC in dimethylsulfoxide. The mixture is agitated at room temperature for 6 hr. Theoligonucleotide is separated from free FITC by elution from a 1×30 cmSephadex G-25 column with 20 mM ammonium acetate (pH 6), combiningfractions in the first UV-absorbing peak.

In general, fluorescent labelling of an oligonucleotide at it's 5′-endinitially involved two steps. First, a N-protected aminoalkylphosphoramidite derivative is added to the 5′-end of an oligonucleotideduring automated DNA synthesis. After removal of all protecting groups,the NHS ester of an appropriate fluorescent dye is coupled to the5′-amino group overnight followed by purification of the labelledoligonucleotide from the excess of dye using reverse phase HPLC or PAGE.

Schubert et al. (1990) described the synthesis of a phosphoramidite thatenables oligonucleotides labeled with fluorescein to be produced duringautomated DNA synthesis. Fluorescein methylester is alkylated with4-chloro(4,4′-dimethoxytrityl)butanol-1 in the presence of K₂CO₃ and KIin DMF for 17 hrs. After removal of the trityl group with 1% TFA inchloroform, the product is phosphitylated by standard procedures withbis(diisopropylamino)methoxyphosphine. Phosphorylation of the aboveobtained fluorescein derivative leads an H-phosphonate in reasonableyields. The resulting amidite (0.1 M solution in dry acetonitrile) isused for the automated synthesis of different primers using β-cyanoethylphosphoramidite chemistry and a DNA synthesizer. Cleavage from thesupport and deprotection is performed with 25% aqueous ammonia for 36hrs at room temperature. The crude product is purified by PAGE and thelabelled primer is visible as a pale green fluorescent band at 310 nm.Elution and desalting using RP 18 cartridges yields the desired product.

The fluorescent labelling of the 5′-end of a probe in the Schubertmethod is directly achieved during DNA synthesis in the last couplingcycle. Coupling yields are as high as with the normal phosphoramidites.After deprotection and removal of ammonia by lyophilization using aspeed vac or by ethanol precipitation, fluorescent labelledoligonucleotides can be directly used for DNA sequencing in Format 3SBH.

Murakami et al. also described the preparation of fluorescein-labeledoligonucleotides. This synthesis is based on a polymer-supportedphosphoramidite and hydrogen phosphonate method. Ethylenediamine orhexamethylenediamine is used as a tether. They were introduced via aphosphoramidate linkage, which was formed by oxidation of ahydrogen-phosphonate intermediate in CCI₄ solution. The modifiedoligonucleotides are subjected to labeling using a primary amineorienting reagent, FITC, on the beads. The resulting modifiedoligonucleotide is cleaved from beads and subsequently purified by RPLC.

Cate et al. (1991) describe the use of oligonucleotide probes directlyconjugated to alkaline phosphatase in combination with a directchemiluminescent substrate (AMPPD) to allow probe detection. Alkalinephosphatase may be covalently coupled to a modified base of theoligonucleotide. After hybridization, the oligo would be incubated withAMPDD. The alkaline phosphatase enzyme breaks AMPDD to yield a compoundthat produces fluorescence without excitation, i.e., a laser is notneeded. It is contemplated that a strong signal can be generated usingsuch technology.

Labelled probes could readily be purchased from a variety of commercialsources, including GEN_(S)ET, rather than synthesized.

EXAMPLE VI Removal of Phosphate Groups

Both bacterial alkaline phosphatase (BAP) and calf intestinal alkalinephosphatase (CIP) catalyze the removal of 5′-phosphate residues from DNAand RNA. They are therefore appropriate for removing 5′ phosphates fromDNA and/or RNA to prevent ligation and inappropriate hybridization.Phosphate removal, as described by Sambrook et al. (1989), would beperformed after cutting, or otherwise shearing, the genomic DNA.

BAP is the more active of the two alkaline phosphatases, but it is alsofar more resistant to heat and detergents. It is therefore difficult toinhibit BAP completely at the end of dephosphorylation reactions.Proteinase K is used to digest CIP, which must be completely removed ifsubsequent ligations are to work efficiently. An alternative method isto inactivate the CIP by heating to 65° C. for 1 hour (or 75° C. for 10minutes) in the presence of 5 mM EDTA (pH 8.0) and then to purify thedephosphorylated DNA by extraction with phenol:chloroform.

EXAMPLE VII Conducting Sequencing by Two Step Hybridization

Following are certain examples to describe the execution of thesequencing methodology contemplated by the inventor. First, the wholechip would be hybridized with mixture of DNA as complex as 100 millionof bp (one human chromosome). Guidelines for conducting hybridizationcan be found in papers such as Drmanac et al. (1990); Khrapko et al.(1991); and Broude et al. (1994). These articles teach the ranges ofhybridization temperatures, buffers and washing steps that areappropriate for use in the initial step of Format 3 SBH.

The present inventor particularly contemplates that hybridization is tobe carried out for up to several hours in high salt concentrations at alow temperature (−2° C. to 5° C.) because of a relatively lowconcentration of target DNA that can be provided. For this purpose, SSCbuffer is used instead of sodium phosphate buffer (Drmanac et al.,1990), which precipitates at 10° C. Washing does not have to beextensive (a few minutes) because of the second step, and can becompletely eliminated when the hybridization cycling is used for thesequencing of highly complex DNA samples. The same buffer is used forhybridization and washing steps to be able to continue with the secondhybridization step with labeled probes.

After proper washing using a simple robotic device on each array, e.g.,a 8×8 mm array (Example III), one labeled, probe, e.g., a 6-mer, wouldbe added. A 96-tip or 96-pin device would be used, performing this in 42operations. Again, a range of discriminatory conditions could beemployed, as previously described in the scientific literature.

The present inventor particularly contemplates the use of the followingconditions. First, after adding labeled probes and incubating forseveral minutes only (because of the high concentration of addedoligonucleotides) at a low temperature (0-5° C.), the temperature isincreased to 3-10° C., depending on F+P length, and the washing bufferis added. At this time, the washing buffer used is one compatible withany ligation reaction (e.g., 100 mM salt concentration range). Afteradding ligase, the temperature is increased again to 15-37° C. to allowfast ligation (less than 30 min) and further discrimination of fullmatch and mismatch hybrids.

The use of cationic detergents is also contemplated for use in Format 3SBH, as described by Pontius & Berg (1991, incorporated herein byreference). These authors describe the use of two simple cationicdetergents, dedecyl- and cetyltrimethylammonium bromide (DTAB and CTAB)in DNA renaturation.

DTAB and CTAB are variants of the quaternary amine tetramethylammoniumbromide (TMAB) in which one of the methyl groups is replaced by either a12-carbon (DTAB) or a 16-carbon (CTAB) alkyl group. TMAB is the bromidesalt of the tetramethylammonium ion, a reagent used in nucleic acidrenaturation experiments to decrease the G-C-content bias of the meltingtemperature. DTAB and CTAB are similar in structure to sodium dodecylsulfate (SDS), with the replacement of the negatively charged sulfate ofSDS by a positively charged quaternary amine. While SDS is commonly usedin hybridization buffers to reduce nonspecific binding and inhibitnucleases, it does not greatly affect the rate of renaturation.

When using a ligation process, the enzyme could be added with thelabeled probes or after the proper washing step to reduce thebackground.

Although not previously proposed for use in any SBH method, ligasetechnology is well established within the field of molecular biology.For example, Hood and colleagues described a ligase-mediated genedetection technique (Landegren et al., 1988), the methodology of whichcan be readily adapted for use in Format 3 SBH. Landegren et al.describe an assay for the presence of given DNA sequences based on theability of two oligonucleotides to anneal immediately adjacent to eachother on a complementary target DNA molecule. The two oligonucleotidesare then joined covalently by the action of a DNA ligase, provided thatthe nucleotides at the junction are correctly base-paired. Although notpreviously contemplated, this situation now arises in Format 3sequencing. Wu & Wallace also describe the use of bacteriophage T4 DNAligase to join two adjacent, short synthetic oligonucleotides. Theiroligo ligation reactions were carried out in 50 mM Tris HCl pH 7.6, 10mM MgCl₂, 1 mM ATP, 1 mM DTT, and 5% PEG. Ligation reactions were heatedto 100° C. for 5-10 min followed by cooling to 0° C. prior to theaddition of T4 DNA ligase (1 unit; Bethesda Research Laboratory). Mostligation reactions were carried out at 30° C. and terminated by heatingto 100° C. for 5 min.

Final washing appropriate for discriminating detection of hybridizedadjacent, or ligated, oligonucleotides of length (F+P), is thenperformed. This washing step is done in water for several minutes at40-60° C. to wash out all the non ligated labeled probes, and all othercompounds, to maximally reduce background. Because of the covalentlybound labeled oligonucleotides, detection is simplified (it does nothave time and low temperature constrains).

Depending on the label used, imaging of the chips is done with differentapparati. For radioactive labels, phosphor storage screen technology andPhosphorImager as a scanner may be used (Molecular Dynamics, Sunnyvale,Calif.). Chips are put in a cassette and covered by a phosphorousscreen. After 1-4 hours of exposure, the screen is scanned and the imagefile stored at a computer hard disc. For the detection of fluorescentlabels, CCD cameras and epifluorescent or confocal microscopy are used.For the chips generated directly on the pixels of a CCD camera,detection can be performed as described by Eggers et al. (1994,incorporated herein by reference).

Charge-coupled device (CCD) detectors serve as active solid supportsthat quantitatively detect and image the distribution of labeled targetmolecules in probe-based assays. These devices use the inherentcharacteristics of microelectronics that accommodate highly parallelassays, ultrasensitive detection, high throughput, integrated dataacquisition and computation. Eggers et al. (1994) describe CCDs for usewith probe-based assays, such as Format 3 SBH of the present invention,that allow quantitative assessment within seconds due to the highsensitivity and direct coupling employed.

The integrated CCD detection approach enables the detection of molecularbinding events on chips. The detector rapidly generates atwo-dimensional pattern that uniquely characterizes the sample. In thespecific operation of the CCD-based molecular detector, distinctbiological probes are immobilized directly on the pixels of a CCD or canbe attached to a disposable cover slip placed on the CCD surface. Thesample molecules can be labeled with radioisotope, chemiluminescent orfluorescent tags.

Upon exposure of the sample to the CCD-based probe array, photons orradioisotope decay products are emitted at the pixel locations where thesample has bound, in the case of Format 3, to two complementary probes.In turn, electron-hole pairs are generated in the silicon when thecharged particles, or radiation from the labeled sample, are incident onthe CCD gates. Electrons are then collected beneath adjacent CCD gatesand sequentially read out on a display module. The number ofphotoelectrons generated at each pixel is directly proportional to thenumber of molecular binding events in such proximity. Consequently,molecular binding can be quantitatively determined (Eggers et al.,1994).

As recently reported, silicon-based CCDs have advantages as solid-statedetection and imaging sensors primarily because of the high sensitivityof the devices over a wide wavelength range (from 1 to 10000 Å). Siliconis very responsive to electromagnetic radiation from the visiblespectrum to soft X-rays. For visible light, a single photon incident onthe CCD gate results in a single electron charge packet beneath thegate. A single soft X-ray beta particle (typically KeV to MeV range)generates thousands to tens of thousands of electrons. In addition tothe high sensitivity, the CCDs described by Eggers et al. (1994) offer awide dynamic range (4 to 5 orders of magnitude) since a detectablecharge packet can range from a few to 10⁵ electrons. The detectionresponse is linear over a wide dynamic range.

By placing the imaging array in proximity to the sample, the collectionefficiency is improved by a factor of at least 10 over lens-basedtechniques such as those found in conventional CCD cameras. That is, thesample (emitter) is in near contact with the detector (imaging array),and this eliminates conventional imaging optics such as lenses andmirrors.

When radioisotopes are attached as reporter groups to the targetmolecules, energetic particles are detected. Several reporter groupsthat emit particles of varying energies have been successfully utilizedwith the micro-fabricated detectors, including ³²P, ³³P, ³⁵S, ¹⁴C and¹²⁵L. The higher energy particles, such as from ³²P, provide the highestmolecular detection sensitivity, whereas the lower energy particles,such as from ³⁵S, provide better resolution. Hence, the choice of theradioisotope reporter can be tailored as required. Once the particularradioisotope label is selected, the detection performance can bepredicted by calculating the signal-to-noise ratio (SNR), as describedby Eggers et al. (1994).

An alternative luminescent detection procedure involves the use offluorescent or chemiluminescent reporter groups attached to the targetmolecules. The fluorescent labels can be attached covalently or throughinteraction. Fluorescent dyes, such as ethidium bromide, with intenseabsorption bands in the near UV (300-350 nm) range and principalemission bands in the visible (500-650 nm) range, are most suited forthe CCD devices employed since the quantum efficiency is several ordersof magnitude lower at the excitation wavelength than at the fluorescentsignal wavelength.

From the perspective of detecting luminescence, the polysilicon CCDgates have the built-in capacity to filter away the contribution ofincident light in the UV range, yet are very sensitive to the visibleluminescence generated by the fluorescent reporter groups. Suchinherently large discrimination against UV excitation enables large SNRs(greater than 100) to be achieved by the CCDs as formulated in theincorporated paper by Eggers et al. (1994).

For probe immobilization on the detector, hybridization matrices may beproduced on inexpensive SiO₂ wafers, which are subsequently placed onthe surface of the CCD following hybridization and drying. This formatis economically efficient since the hybridization of the DNA isconducted on inexpensive disposable SiO₂ wafers, thus allowing reuse ofthe more expensive CCD detector. Alternatively, the probes can beimmobilized directly on the CCD to create a dedicated probe matrix.

To immobilize probes upon the SiO₂ coating, a uniform epoxide layer islinked to the film surface, employing an epoxy-silane reagent andstandard SiO₂ modification chemistry. Amine-modified oligonucleotideprobes are then linked to the SiO₂ surface by means of secondary amineformation with the epoxide ring. The resulting linkage provides 17rotatable bonds of separation between the 3′ base of the oligonucleotideand the SiO₂ surface. To ensure complete amine deprotonation and tominimize secondary structure formation during coupling, the reaction isperformed in 0.1 M KOH and incubated at 37° C. for 6 hours.

In Format 3 SBH in general, signals are scored per each of billionpoints. It would not be necessary to hybridize all arrays, e.g., 40005×5 mm, at a time and the successive use of smaller number of arrays ispossible.

Cycling hybridizations are one possible method for increasing thehybridization signal. In one cycle, most of the fixed probes willhybridize with DNA fragments with tail sequences non-complementary forlabelled probes. By increasing the temperature, those hybrids will bemelted (FIG. 3). In the next cycle, some of them (˜0.1%) will hybridizewith an appropriate DNA fragment and additional labeled probes will beligated. In this case, there occurs a discriminative melting of DNAhybrids with mismatches for both probe sets simultaneously.

In the cycle hybridization, all components are added before the cyclingstarts, at the 37° C. for T4, or a higher temperature for a thermostableligase. Then the temperature is decreased to 15-37° C. and the chip isincubated for up to 10 minutes, and then the temperature is increased to37° C. or higher for a few minutes and then again reduced. Cycles can berepeated up to 10 times. In one variant, an optimal higher temperature(10-50° C.) can be used without cycling and longer ligation reaction canbe performed (1-3 hours).

The procedure described herein allows complex chip manufacturing usingstandard synthesis and precise spotting of oligonucleotides because arelatively small number of oligonucleotides are necessary. For exampleif all 7-mer oligos are synthesized (16384 probes), lists of 256 million14-mers can be determined.

One important variant of the invented method is to use more than onedifferently labeled probe per basic array. This can be executed with twopurposes in mind; multiplexing to reduce number of separately hybridizedarrays; or to determine a list of even longer oligosequences such as 3×6or 3×7. In this case if two labels are used the specificity of the 3consecutive oligonucleotides can be almost absolute because positivesites must have enough signals of both labels.

A further and additional variant is to use chips containing BxNy probeswith y being from 1 to 4. Those chips allow sequence reading indifferent frames. This can also be achieved by using appropriate sets oflabeled probes or both F and P probes could have some unspecified endpositions (i.e., some element of terminal degeneracy). Universal basesmay also be employed as part of a linker to join the probes of definedsequence to the solid support. This makes the probe more available tohybridization and makes the construct more stable. If a probe has 5bases, one may, e.g., use 3 universal bases as a linker (FIG. 4).

EXAMPLE VIII Analyzing the Data Obtained

Image files are analyzed by an image analysis program, like DOTS program(Drmanac et al., 1993), and scaled and evaluated by statisticalfunctions included, e.g., in SCORES program (Drmanac et al., 1994). Fromthe distribution of the signals an optimal threshold is determined fortransforming signal into +/− output.

From the position of the label detected, F+P nucleotide sequences fromthe fragments would be determined by combining the known sequences ofthe immobilized and labelled probes corresponding to the labelledpositions. The complete nucleic acid sequence or sequence subfragmentsof the original molecule, such as a human chromosome, would then beassembled from the overlapping F+P sequences determined by computationaldeduction.

One option is to transform hybridization signals e.g., scores, into +/−output during the sequence assembly process. In this case, assembly willstart with a F+P sequence with a very high score, for example F+Psequence AAAAAATTTTTT (SEQ ID NO:1). Scores of all four possibleoverlapping probes AAAAATTTTTTA (SEQ ID NO:3), AAAAATTTTTTT (SEQ IDNO:4), AAAAATTTTTTC (SEQ ID NO:5) and AAAAATTTTTTG (SEQ ID NO:6) andthree additional probes that are different at the beginning(TAAAAATTTTTT, SEQ ID NO:7; CAAAAATTTTTT, SEQ ID NO:8; GAAAAATTTTTT, SEQID NO:9) are compared and three outcomes defined: (i) only the startingprobe and only one of the four overlapping probes have scores that aresignificantly positive relatively to the other six probes, in this casethe AAAAAATTTTTT (SEQ ID NO:1) sequence will be extended for onenucleotides to the right; (ii) no one probe except the starting probehas a significantly positive score, assembly will stop, e.g., theAAAAAATTTTT (SEQ ID NO:10) sequence is at the end of the DNA moleculethat is sequenced; (iii) more than one significantly positive probeamong the overlapped and/or other three probes is found; assembly isstopped because of the error or branching (Drmanac et al., 1989).

The processes of computational deduction would employ computer programsusing existing algorithms (see, e.g., Pevzner, 1989; Drmanac et al.,1991; Labat and Drmanac, 1993; each incorporated herein by reference).

If, in addition to F+P, F(space 1)P, F(space 2)P, F(space 3)P or F(space4)P are determined, algorithms will be used to match all data sets tocorrect potential errors or to solve the situation where there is abranching problem (see, e.g., Drmanac et al., 1989; Bains et al., 1988;each incorporated herein by reference).

EXAMPLE IX Re-using Sequencing Chips

When ligation is employed in the sequencing process, then the ordinaryoligonucleotides chip cannot be immediately reused. The inventorcontemplates that this may be overcome in various ways.

One may employ ribonucleotides for the second probe, probe P, so thatthis probe may subsequently be removed by RNAase treatment. RNAasetreatment may utilize RNAase A an endoribonuclease that specificallyattacks single-stranded RNA 3′ to pyrimidine residues and cleaves thephosphate linkage to the adjacent nucleotide. The end products arepyrimidine 3′ phosphates and oligonucleotides with terminal pyrimidine3′ phosphates. RNAase A works in the absence of cofactors and divalentcations.

To utilize an RNAase, one would generally incubate the chip in anyappropriate RNAase-containing buffer, as described by Sambrook et al.(1989; incorporated herein by reference). The use of 30-50 μl ofRNAase-containing buffer per 8×8 mm or 9×9 mm array at 37° C. forbetween 10 and 60 minutes is appropriate. One would then wash withhybridization buffer.

Although not widely applicable, one could also use the uracil base, asdescribed by Craig et al. (1989), incorporated herein by reference, inspecific embodiments. Destruction of the ligated probe combination, toyield a re-usable chip, would be achieved by digestion with the E. colirepair enzyme, uraci-DNA glycosylase which removes uracil from DNA.

One could also generate a specifically cleavable bond between the probesand then cleave the bond after detection. For example, this may achievedby chemical ligation as described by Shabarova et al. (1991) andDolinnaya et al. (1988), both references being specifically incorporatedherein by reference.

Shabarova et al. (1991) describe the condensation of oligodeoxyribonucleotides with cyanogen bromide as a condensing agent. In their onestep chemical ligation reaction, the oligonucleotides are heated to 97°C., slowly cooled to 0° C., then 1 μl 10M BrCN in acetonitrile is added.

Dolinnaya et al. (1988) show how to incorporate phosphoramidate andpyrophosphate internucleotide bonds in DNA duplexes. They also use achemical ligation method for modification of the sugar phosphatebackbone of DNA, with a water-soluble carbodiimide (CDI) as a couplingagent. The selective cleavage of a phosphoamide bonds involves contactwith 15% CH₃COOH for 5 min at 95° C. The selective cleavage of apyrophosphate bond involves contact with a pyridine-water mixture (9:1)and freshly distilled (CF₃CO)₂O.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations may be applied to the composition, methodsand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentsthat are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims. All claimedmatter and methods can be made and executed without undueexperimentation.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method of determining a nucleotide sequence of a target nucleicacid, comprising the steps of: (a) contacting a target nucleic acid witha set of immobilized oligonucleotide probe(s) and at least one labeledoligonucleotide probe from a set of labeled oligonucleotide probes underhybridization conditions effective to permit hybridization between: (i)complementary sequences of the target nucleic acid and the immobilizedprobes and (ii) complementary sequences of the target nucleic acid andthe labeled probe(s); (b) covalently joining immobilized probe(s) andlabeled probe(s) which are adjacently hybridized to the same targetnucleic acid molecule; (c) detecting the labels of the labeledoligonucleotide probe(s) that are covalently joined to the immobilizedprobe(s); (d) identifying at least one nucleotide sequence in the targetnucleic acid by steps comprising connecting the nucleotide sequences ofthe detected labeled oligonucleotide probe(s) with the nucleotidesequences of their respective joined immobilized oligonucleotideprobe(s) (e) analyzing the sequences identified in step (d) to determineidentified sequences that overlap and (f) reconstructing said sequencefrom overlapping oligonucleotide sequences identified in step (e).
 2. Amethod according to claim 1 wherein the set of immobilizedoligonucleotide probes comprises a first set of immobilizedoligonucleotide probes and the at least one labeled oligonucleotideprobe comprises a first labeled oligonucleotide probe, the methodfurther comprising the step of, between steps a) and b): contacting thetarget nucleic acid with a second set of immobilized oligonucleotideprobe(s) and a second labeled oligonucleotide probe under hybridizationconditions effective to permit hybridization between: (i) complementarysequences of the target nucleic acid and the immobilized probes of thesecond set and (ii) complementary sequences of the target nucleic acidand the second labeled oligonucleotide probe, wherein the first andsecond labeled oligonucleotide probes comprise different nucleotidesequences and the labels of the first and second labeled oligonucletideprobes are the same.
 3. The method of claim 1 wherein said labels aredetected in situ.
 4. The method of claim 1, wherein said covalentlyjoining immobilized probe(s) and labeled probe(s) comprises contactingsaid probes with a ligating agent.
 5. The method of claim 4 wherein saidlabeled probe(s) are contacted with the target nucleic acid at the sametime as said ligating agent.
 6. The method of claim 1, wherein afterstep (b) and before step (c), labeled probes that are not covalentlyjoined to an immobilized probe are removed.
 7. The method of claim 1 inwhich labeled probes that are not covalently joined to an immobilizedprobe are removed under stringent washing conditions.
 8. The method ofclaim 1 in which a plurality of immobilized probes are immobilized onthe same support.
 9. The method of claim 1 in which immobilized probeshaving different nucleotide sequences are immobilized on differentsupports.
 10. The method of claim 1 in which the immobilizedoligonucleotide probes comprises a plurality of arrays arranged in theform of a sequencing chip.
 11. The method of claim 2 in which aplurality of immobilized probes of the first set are immobilized on asupport and/or a plurality of immobilized probes of the second set areimmobilized on the same support.
 12. The method of claim 11 in which thefirst and second sets of immobilized probes comprise a sequencing chip.13. The method of claim 2 in which immobilized probes of the first sethaving different nucleotide sequences are immobilized on differentsupports and/or immobilized probes of the second set having differentnucleotide sequences are immobilized on different supports.
 14. Themethod of claim 1 in which in step (a), the target nucleic acid iscontacted with a set of labeled oligonucleotide probes in a sequentialmanner, one labeled oligonucleotide probe at a time.
 15. The method ofclaim 14 in which in step (a), labeled oligonucleotide probes of the setwhich have different nucleotide sequences are labeled with the samelabel.
 16. The method of claim 14 in which in step (a), at least twolabeled oligonucleotide probes of the set which have differentnucleotide sequences are labeled with different labels.
 17. The methodof claim 14 in which in step (a), the target nucleic acid is contactedsimultaneously with the set of immobilized probes and the labeledoligonucleotide probe.
 18. The method of claim 14 in which in step (a),the target nucleic acid is contacted first with the set of immobilizedprobes to form immobilized probe:target complexes and thereafter withthe labeled oligonucleotide probe.
 19. The method of claim 1 in which instep (a), the target nucleic acid is contacted simultaneously with atleast two labeled oligonucleotide probes of a set of labeledoligonucleotide probes, wherein said at least two labeledoligonucleotide probes are labeled with different, distinguishablelabels and have different nucleotide sequences that are identifiable bythe properties of their respective labels.
 20. The method of claim 19 inwhich in step (a), the target nucleic acid is contacted simultaneouslywith the set of immobilized probes and said at least two labeledoligonucleotide probes.
 21. The method of claim 19 in which in step (a),the target nucleic acid is contacted first with the set of immobilizedprobes to form immobilized probe:target complexes and thereafter withsaid at least two labeled oligonucleotide probes.
 22. The method ofclaim 1 in which in step (a), the target nucleic acid is contactedsimultaneously with at least two labeled oligonucleotide probes of a setof labeled oligonucleotide probes, wherein said at least two labeledoligonucleotide probes are labeled with different, distinguishablelabels and have different nucleotide sequences that are identifiable bythe properties of their respective labels and in step (d) the nucleotidesequences of the immobilized and labeled probes are determined byobserving in situ the properties of the labels and their relativepositions within an array.
 23. The method of claim 22 in which in step(a), the target nucleic acid is contacted simultaneously with the arrayof immobilized probes and said at least two labeled oligonucleotideprobes.
 24. The method of claim 22 in which in step (a), the targetnucleic acid is contacted first with the array of immobilized probes toform immobilized probe:target complexes and thereafter with said atleast two labeled oligonucleotide probes.
 25. The method of claim 1,wherein the complete nucleotidle sequence of the target nucleic acid isdetermined.
 26. The method of claim 1, wherein the target nucleic acidis mapped.
 27. The method of claim 1, wherein the target nucleic acid ispartially sequenced.
 28. The method of claim 1, wherein the immobilizedoligonucleotide probes have a length F and the labeled oligonucleotideprobes have a length P, where F and P are each independently between 4and 9 nucleotides.
 29. The method of claim 1 wherein said immobilizedoligonucleotide probe(s) and/or said labeled probe(s) additionallycomprise a universal base or all four bases at the terminal positionthereof.
 30. The method of claim 1 wherein the target nucleic acid isfragmented prior to step (a).
 31. The method of claim 30 wherein thetarget nucleic acid is fragmented by restriction enzyme digestion,ultrasound treatment, NaOH treatment or low pressure shearing.
 32. Themethod of claim 30 wherein the target nucleic acid fragments have alength T, the immobilized oligonucleotide probes have a length F and thelabeled oligonucleotide probes have a length P, where T is between 10and 100 nucleotides and F and P are each independently between 4 and 9nucleotides.
 33. The method of claim 32 wherein T is between 10 and 40nucleotides.
 34. The method of claim 32 wherein T is about 20nucleotides.
 35. The method of claim 32 wherein T is about 3 timeslonger than F.
 36. The method of any one of claims 32 through 35 whereinF and P are each 6 nucleotides.
 37. The method of claim 1 wherein theadjacently hybridized immobilized and labeled probe(s) are covalentlyjoined to one another by enzymatic ligation.
 38. The method of any ofclaims 1 or 2 wherein the hybridization is carried out in cycles. 39.The method of any one of claims 1 or 2 wherein the hybridizationconditions are effective to permit hybridization between target nucleicacids and only those immobilized probes and labeled probes that areperfectly complementary to a portion of the target.
 40. The method ofany one of claims 1 or 2 wherein the hybridization conditions areeffective to permit hybridization between those immobilized probes andlabeled probes that are immediately adjacent to each other and hybridizeto the same target nucleic acid molecule.
 41. The method of any one ofclaims 1 or 2 wherein the target nucleic acid is a cloned DNA, achromosomal DNA or a mRNA.
 42. The method of any one of claims 1 or 2wherein the immobilized oligonucleotide probes are immobilized by way ofcovalent attachment.
 43. The method of claim 42 wherein the immobilizedprobes are immobilized via a phosphodiester linkage.
 44. The method ofclaim 42 wherein the immobilized probes are immobilized via a linker.45. The method of any one of claims 1 or 2 wherein the immobilizedprobes are immobilized on glass, polystyrene or teflon.
 46. The methodof any one of claims 1 or 2 wherein the label is a radioactive isotope,non-radioactive isotope or a moiety that emits light.
 47. The method ofclaim 42 wherein the label is a fluorescent dye.
 48. The method of anyone of claims 1 or 2 wherein the target nucleic acid, an immobilizedprobe or a labeled probe comprises a modified base or a universal base.49. The method of any one of claims 1 or 2 wherein the immobilized probecontains a modification to allow the immobilized probe to be reusedafter said hybridization.
 50. The method of claim 49 wherein theoligonucleotides of the labeled probe comprise ribonucleotides.
 51. Themethod of claim 50 wherein said covalently joined labeled probecomprising ribonucleotides is removed from the immobilized probe byRNAase treatment.
 52. The method of claim 49 wherein the covalentlyjoined labeled probe comprises a uracil base.
 53. The method of claim 52wherein said covalently joined labeled probe comprising a uracil base isremoved from the immobilized probe by uracil-DNA glycosylase treatment.54. The method of claim 49 wherein said labeled probe comprises achemically cleavable bond.